The invention relates to the fields of molecular genetics and pharmacology. The invention provides methods and compositions for determining the capability of a compound, a macromolecular species, or of a stimulatory effector to produce a conformational change in a predetermined nuclear receptor and/or its accessory protein(s), typically to agonize or antagonize a ligand-induced activation of the nuclear receptor. An aspect of the invention can provide a means for identifying agents which are pharmacological agonists or antagonists for one or more predetermined nuclear receptor species. An aspect of the invention relates to a method of rank-ordering a set of compounds with respect to each compound""s ability to affect an interaction between one or more nuclear receptor species and a plurality of coactivator and/or corepressor species or similar interfaces; the rank-ordering provides a dataset for identifying pharmacologically active agonists, antagonists, partial agonists, potentiators, and the like.
A variety of nuclear receptors exist in animal cells and generally function to effectuate transcriptional regulation on one or more subsets of regulable genes. Most often, the nuclear receptor exhibits a high-affinity binding interaction with one or more species of hydrophobic ligand. These ligand binding interactions can produce a conformational change in the nuclear receptor. The conformational change induced by ligand binding modifies the ability of the nuclear receptor to interact with certain specific receptor-binding DNA sequences and/or to interact with other nuclear proteins (e.g., transcription factors, coactivators, corepressors), so as to modulate the transcription of genes having the specific receptor-binding DNA sequence(s) located so as to influence the transcription of the gene. In one model of steroid receptor action, a ligand-induced conformational change in a ligand-binding domain unmasks a DNA-binding activity in another structural domain of the steroid receptor protein. In the absence of this ligand-induced conformational change, the ligand-binding domain represses the DNA binding activity of the linked structural domain. It has been recently shown that one or more superfamily of proteins, termed xe2x80x9ccoactivatorsxe2x80x9d, and xe2x80x9ccorepressorsxe2x80x9d, respectively interact with nuclear receptors in a ligand-dependent fashion so as to effect transcriptional activation (coactivators) or so as to inhibit or silence transcription (corepressors) of genes which are transcriptionally modulated by nuclear receptors.
Nuclear hormone receptors comprise a superfamily of over 40 transcription actors. About half of them are classical receptors for lipophilic ligands such as steroids and vitamins. The nuclear hormone receptor gene superfamily encodes structurally related proteins that regulate transcription of target genes. These macromolecules include receptors for steroid and thyroid hormones, vitamins, retinoids, fatty acids, and other nuclear receptor proteins for which no ligands have been found, so-called xe2x80x9corphan receptorsxe2x80x9d. These receptors have modular domains with readily identifiable structural features and sequence motifs. The DNA-binding domain (xe2x80x9cDBDxe2x80x9d) directs the receptors to bind specific DNA sequences as monomers, homodimers, or heterodimers. The ligand-binding domain (xe2x80x9cLBDxe2x80x9d) responds to binding of the cognate hormone; this domain and the amino terminal domain interact with other transcription factors, and with the coactivators and/or corepressors. Nuclear receptor-specific actions are derived from a combination of diverse elements, including availability of ligand, receptors, and nonreceptor factors; target-site structure; interactions with other proteins, such as the general transcription factors and very importantly with the coactivator and/or corepressor proteins.
The steroid/thyroid hormone receptor superfamily of ligand-activated transcription factors encompasses not only the receptors for steroids, thyroid hormone, retinoids and vitamin D, but also a large number of proteins whose functions and/or ligands are unknown and which are thus termed orphan receptors. This family of transcription factors integrates signals from ligands as well as from signal transduction pathways, resulting in alterations in mRNA and protein expression that are unique to the complex signals received. These nuclear receptors are implicated in the control of a wide range of physiological responses and homeostatic conditions, including cell differentiation, neoplasia, control of cellular metabolism, and neurological function. For a review of the steroid hormone receptor superfamily, see Ribiero R C (1995) Annu. Rev. Med. 46: 443-453.
There has been substantial interest in identifying ligands which interact with nuclear receptors and modulate the biological effects mediated by these nuclear receptors. Such ligands, whether agonistic or antagonistic to natural physiological ligands of the receptors, would serve as candidate pharmaceuticals for controlling the biological effects of nuclear receptor-mediated transcriptional control and the attendant physiological effects produced thereby. Unfortunately, most conventional assays for identifying potential ligands rely upon the use of libraries of radiolabeled compounds which are tested for their binding coefficient (e.g., via Scatchard analysis) to a purified nuclear receptor species. It is difficult and labor-intensive to obtain such libaries of radiolabeled compounds and then screen the library using binding assays. Furthermore, it has been found that a compound""s binding constant is not necessarily predictive of its biological activity as a ligand. As a better proxy for ligand function, transcriptional assays have been developed to assay for ligand-induced transcriptional activation of a nuclear receptor as detected by transcription of a reporter sequence operably linked to a nuclear receptor response element and promoter.
Unfortunately, many of the transcriptional responses generated by ligand-activated nuclear receptors can be subtle and are frequently difficult to detect and/or quantify by conventional transcriptional assay procedures, which are relatively insensitive for monitoring expression of genes which are not abundantly transcribed. Furthermore, many of the conventional transcription assay procedures are difficult to perform and entail problematic steps, such as requiring lysis of the cells being assayed. It is desirable to have a method for detecting ligands of predetermined nuclear receptors with high specificity, sensitivity, and selectivity. In particular, methods to reduce readout background noise that can obscure legitimate signals would find great use in the art for identifying novel pharmaceutical agents that are nuclear receptor ligands, as well as providing sensitive assays for detection and quantitation of ligands which are environmental pollutants that activate nuclear receptors (e.g., TCDD).
Moreover, many nuclear receptor ligands, particularly steroids and steroid-like compounds, often exhibit pleiotropic biological effects through nuclear receptors. For example, both estradiol and tamoxiphen bind to the estrogen receptor, but each compound can produce different biological effects and transcriptional profiles (i.e., the set of genes which are transcriptionally modulated by ligand presence) depending upon the tissue and cell-type involved.
Thus, there exists a need in the art for methods to efficiently identify agents which modulate nuclear receptor function. It is important that such methods have the necessary levels of sensitivity and specificity for identifying bona fide nuclear receptor agonists and/or antagonists. The present invention fulfills these and other needs in the art.
The references discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention.
In accordance with the foregoing, in one aspect of the invention are provided methods for identifying agents which are agonists or antagonists for ligand-induced activation of a predetermined nuclear receptor. There are a variety of formats of the invention, and a plurality of formats may be multiplexed sequentially or in parallel to produce a discrimination profile of a test agent in order to categorize the pharmacological nature of the test agent (e.g., agonist, antagonist, partial agonist, mixed agonist/antagonist, etc.), so as to enable dissecting the pleotropic biological effects of nuclear receptor ligands and correlating the individual effects with chemical structure.
The methods of the invention typically employ a positive hybrid nuclear receptor signal transduction system, which typically comprises an intact eukaryotic host cell, comprising: (1) a LBD-TRX polynucleotide sequence encoding and expressing a ligand-activatable fusion protein (referred to herein as xe2x80x9cLBD-TRXxe2x80x9d) which comprises a ligand binding domain (xe2x80x9cLBDxe2x80x9d) of a predetermined nuclear receptor covalently linked, typically in polypeptide linkage, to a transcriptional activator domain (xe2x80x9cADxe2x80x9d) or alternatively to a DNA-binding domain (xe2x80x9cDBDxe2x80x9d) of a predetermined transcription factor (generically, AD and DBD are referred to as xe2x80x9cTRXxe2x80x9d for convenience),(2) a CA-TRX polynucleotide sequence encoding and expressing a coactivator fusion protein (referred to herein as xe2x80x9cCA-TRXxe2x80x9d) which comprises a domain of a nuclear receptor coactivator protein (xe2x80x9cCAxe2x80x9d) capable of binding to said LBD of said predetermined nuclear receptor linked, typically in polypeptide linkage, to a transcriptional activator domain or alternatively to a DNA-binding domain of a predetermined transcription factor (collectively xe2x80x9cTRXxe2x80x9d), and (3) a reporter polynucleotide sequence comprising, in linear order, a transcriptional regulatory sequence which is responsive to said predetermined transcription factor and a reporter cassette encoding a sequence that confers a signal or detectable phenotype. The TRX components of the LBD-TRX protein and the CA-TRX protein should be functionally complementary; i.e., if the TRX of one fusion is a DBD, the TRX of the other fusion is an AD, and vice versa. Prior to contacting of the system with an agent which is an agonist ligand of the LBD, the transcriptional activity of the LBD-TRX and CA-TRX with respect to the reporter polynucleotide is substantially absent and the system substantially lacks functional expression of an encoded sequence of the reporter polynucleotide. Subsequent to contacting of the system with an agonist agent which produces an activated conformation of the LBD in the LBD-TRX fusion protein, the CA-TRX fusion protein functionally associates forming a LBD-TRX/CA-TRX complex that is transcriptionally active with respect to the reporter polynucleotide, whereupon the reporter cassette can be transcribed and functionally expressed under the control of the transcriptional regulatory sequence. Thus, functional expression of the reporter cassette serves to report whether the system has been contacted with an agent which induced an activated conformation of the LBD. Advantageously, in one variation, the reporter cassette encodes a cell surface reporter protein which can be detected and/or selected for on the basis of its presence on the surface of a cell membrane. In embodiments where the nuclear receptor signal transduction system consists of metabolically active cells having a polynucleotide sequence encoding the LBD-TRX fusion, the CA-TRX fusion, and a reporter polynucleotide sequence, the system is referred to as a CA-TRX reporter cell.
A nuclear receptor signal transduction system of the present invention can be used to evaluate one or more test agents for their ability to activate the LBD of a predetermined nuclear receptor. Typically, the ability to activate the LBD indicates an agonistic activity as referenced to the physiological ligand of the nuclear receptor. In this aspect of the invention, a test agent is applied to a nuclear receptor signal transduction system and incubated for a suitable incubation period anticipated to be substantially sufficient for the agent to transduce a signal to the LBD. After the incubation period, the expression of the reporter cassette is determined by detecting the presence of the reporter. Test agents which produce a statistically significant increase in reporter as compared to background (e.g., placebo) are scored as receptor agonists. In embodiments where the nuclear receptor signal transduction system comprises a metabolically active intact reporter cell, typically a population of reporter cells is contacted with a test agent, and the ability of the test agent to function as a receptor agonist is determined by either the relative expression of detectable reporter above background as compared to a reference population of reporter cells under substantially equivalent conditions in the absence of said test agent. Dose-response data can be generated in this manner using a variety of different concentrations of the test agent.
In a variation, the method may be used to identify antagonists of a predetermined nuclear receptor LBD by determining the ability of a test agent to produce a statistically significant reduction in the expression the reporter by inhibiting the capacity of a unit dose of a predetermined activating ligand of the LBD from producing the amount of expression of the reporter as occurs in the absence of the test agent.
The invention can also employ a composition and method for identifying ligands which induce binding of a corepressor to the LBD. These systems typically employ a positive hybrid nuclear receptor signal transduction system (a xe2x80x9cCR-TRXxe2x80x9d system), which typically comprises an intact eukaryotic host cell, comprising: (1) a LBD-TRX polynucleotide sequence encoding and expressing a ligand-activatable fusion protein (referred to herein as xe2x80x9cLBD-TRXxe2x80x9d) which comprises a ligand binding domain (xe2x80x9cLBDxe2x80x9d) of a predetermined nuclear receptor covalently linked, typically in polypeptide linkage, to a transcriptional activator domain (xe2x80x9cADxe2x80x9d) or alternatively to a DNA-binding domain (xe2x80x9cDBDxe2x80x9d) of a predetermined transcription factor (generically, AD and DBD are referred to as xe2x80x9cTRXxe2x80x9d for convenience),(2) a CR-TRX polynucleotide sequence encoding and expressing a corepressor fusion protein (referred to herein as xe2x80x9cCR-TRXxe2x80x9d) which comprises a domain of a nuclear receptor corepressor protein (xe2x80x9cCRxe2x80x9d) capable of binding to said LBD of said predetermined nuclear receptor linked, typically in polypeptide linkage, to a transcriptional activator domain or alternatively to a DNA-binding domain of a predetermined transcription factor (collectively xe2x80x9cTRXxe2x80x9d), and (3) a reporter polynucleotide sequence comprising, in linear order, a transcriptional regulatory sequence which is responsive to said predetermined transcription factor and a reporter cassette encoding a sequence that confers a signal or detectable phenotype. The TRX components of the LBD-TRX protein and the CR-TRX protein should be functionally complementary; i.e., if the TRX of one fusion is a DBD, the TRX of the other fusion is an AD, and vice versa. Prior to contacting of the system with an agent which is an angonoist ligand of the LBD, the transcriptional activity of the LBD-TRX and CR-TRX with respect to the reporter polynucleotide is substantially absent and the system substantially lacks functional expression of an encoded sequence of the reporter polynucleotide. Subsequent to contacting of the system with an antagonist agent which produces an activated conformation of the LBD in the LBD-TRX fusion protein, the CR-TRX fusion protein functionally associates forming a LBD-TRX/CR-TRX complex that is transcriptionally active with respect to the reporter polynucleotide, whereupon the reporter cassette can be transcribed and functionally expressed under the control of the transcriptional regulatory sequence. Thus, functional expression of the reporter cassette serves to report whether the system has been contacted with an agent which induced an activated conformation of the LED. Advantageously, in one variation, the reporter cassette encodes a cell surface reporter protein which can be detected and/or selected for on the basis of its presence on the surface of a cell membrane. In embodiments where the nuclear receptor signal transduction system consists of metabolically active cells having a polynucleotide sequence encoding the LBD-TRX fusion, the CR-TRX fusion, and a reporter polynucleotide sequence, the system is referred to as a CR-TRX reporter cell.
A nuclear receptor signal transduction system of the present invention can be used to evaluate one or more test agents for their ability to activate the LBD of a predetermined nuclear receptor and induce corepressor binding. Typically, the ability to activate the LBD in this manner indicates an antagonistic or partial (mixed) antagonistic activity as referenced to a physiological ligand of the nuclear receptor. In this aspect of the invention, a test agent is applied to a CR-TRX system and incubated for a suitable incubation period anticipated to be substantially sufficient for the agent to transduce a signal to the LBD. After the incubation period, the expression of the reporter cassette is determined by detecting the presence of the reporter. Test agents which produce a statistically significant increase in reporter as compared to background (e.g., placebo) are scored as receptor antagonists or partial (mixed) antagonists. In embodiments where the nuclear receptor signal transduction system comprises a metabolically active intact reporter cell, typically a population of reporter cells is contacted with a test agent, and the ability of the test agent to function as a receptor agonist is determined by either the relative expression of detectable reporter above background as compared to a reference population of reporter cells under substantially equivalent conditions in the absence of said test agent. Dose-response data can be generated in this manner using a variety of different concentrations of the test agent.
In a variation, the method may be used to identify agonists or partial (mixed) agonists of a predetermined nuclear receptor LBD by determining the ability of a test agent to produce a statistically significant reduction in the expression the reporter by inhibiting the capacity of a unit dose of a predetermined antagonist ligand of the LBD from producing the amount of expression of the reporter as occurs in the absence of the test agent.
In a variation, the invention provides a nuclear receptor signal transduction system, comprising a xe2x80x9creverse hybridxe2x80x9d reporter host cell containing: (1) an LBD-TRX polynucleotide sequence encoding a fusion protein comprising a ligand-binding domain of a nuclear receptor in polypeptide linkage a DBD or AD of a transcription factor; (2) a CR-TRX polynucleotide sequence encoding and expressing a corepressor fusion protein (referred to herein as xe2x80x9cCR-TRXxe2x80x9d) which comprises a domain of a nuclear receptor corepressor protein (xe2x80x9cCRxe2x80x9d) capable of binding to said LBD of said predetermined nuclear receptor linked, typically in polypeptide linkage, to a transcriptional activator domain or alternatively to a DNA-binding domain of a predetermined transcription factor (TRX), and (3) a relay (or signal inverter) gene encoding a protein which is efficiently expressed as a consequence of the LDB-TRX binding to the CR-TRX as a transcriptionally active complex, and (4) reporter gene comprising, in linear order, a transcriptional regulatory sequence responsive to said transcriptionally active complex, and a reporter cassette encoding a reporter, wherein the reporter gene is efficiently expressed then the product of the relay (or signal inverter) gene is substantially absent and is either poorly expressed or not expressed when the relay (or signal inverter) gene is efficiently expressed. In an aspect, the LBD is a functional portion of a steroid hormone superfamily receptor capable of binding a corepressor protein in the absence of activating ligand and capable of undergoing a stimulus-induced conformational change, such as by binding a known agonistic ligand, so as to reduce or abrogate binding to the corepressor. The reverse hybrid system facilitates identification of test agents that relieve corepressor binding to the LBD.
A reverse hybrid nuclear receptor signal transduction system of the present invention can be used to evaluate one or more test agents for their ability to activate the LBD of a predetermined nuclear receptor by relieving corepressor binding. Typically, the ability to activate the LBD indicates an agonistic activity as referenced to the physiological ligand of the nuclear receptor, however given the pleotropic nature of many nuclear receptor ligands, it is possible that some such agents will be agonistic with respect to certain effects and antagonistic with respect to others. In this aspect of the invention, a test agent is applied to a reverse hybrid nuclear receptor signal transduction system and incubated for a suitable incubation period anticipated to be substantially sufficient for the agent to transduce a signal to the LBD. After the incubation period, the expression of the reporter cassette is determined by detecting the presence of the reporter. Test agents which produce a statistically significant increase in reporter as compared to background (e.g., placebo) are scored as receptor agonists. In embodiments where the reverse hybrid nuclear receptor signal transduction system comprises a metabolically active intact reporter cell, typically a population of reporter cells is contacted with a test agent, and the ability of the test agent to function as a receptor agonist is determined by either the relative expression of detectable reporter above background as compared to a reference population of reporter cells under substantially equivalent conditions in the absence of said test agent. Dose-response data can be generated in this manner using a variety of different concentrations of the test agent.
In a variation, the method may be used to identify antagonists of a predetermined nuclear receptor LBD by determining the ability of a test agent to produce a statistically significant reduction in the expression the reporter by inhibiting the capacity of a unit dose of a predetermined activating ligand of the LBD from producing the amount of expression of the reporter as occurs in the absence of the test agent.
In a variation of the reverse hybrid system, a coactivator (CA) domain may be used instead of a corepressor (CR) domain, and the system can be used, for example, to identify agents that inhibit, abrogate, disrupt, dinimish, interfere with, or otherwise antagonize the functional interaction of the LBD and the coactivator domain. Such agents need not necessarily interact with the ligand-binding pocket of the LBD, and, for example, may interact with a binding interface between the coactivator and the LDB, or at other sites. The method may be used to identify functional antagonists of a predetermined nuclear receptor LBD by determining the ability of a test agent to produce a statistically significant reduction or relative inhibition in the expression the reporter by inhibiting the capacity of a unit dose of a predetermined activating ligand of the LBD from producing the amount of expression of the reporter as occurs in the absence of the test agent.
The invention can also be modified to identify agents that modulate, either positively or negatively, binding interactions between an LBD and a coactivator or corepressor without said agents necessarily interacting with a bona fide ligand-binding domain of the LBD. Generally, these formats employ an LBD, or binding portion thereof, which is functionally constitutive (i.e., not ligand-dependent) for binding to a coactivator or corepressor, or a binding fragment thereof. Such constitutive LBD species can be found as natural variants, can be generated by mutagenesis and selection for constitutive function, or can be generated by employing a high-affinity bona fide ligand at high concentration or covalently attached to the ligand binding pocket (e.g., via photoaffinity labelling), or other means known to those skilled in the art. In such systems, the constitutive LBD and coactivator (or corepressor) are used in LBD-TRX and CA-TRX (or CR-TRX) fusions in a reverse hybrid system and agents are screened for their ability to inhibit the constitutive LBD:CA (or LBD:CR) interaction as measured by a positive readout of the reporter gene.
In a variation of the invention, direct physical interaction, measured as binding, between a LBD domain and a CA domain (or CR domain) as a consequence of ligand presence can be determined. In an aspect, an LBD domain is immobilized on a capture surface and a soluble, labelled or epitope-tagged CA or CR domain is introduced under aqueous physiological conditions, either in the absence or presence of a known ligand or a test agent. A typical format of the direct method can be an ELISA, for illustration. Agents which produce a ligand-induced binding between the immobilized LBD and the soluble, labelled or epitope-tagged CA (or CR), thereby resulting in the CA (or CR) becoming immobilized on the capture surface and retained following washing of the surface with a rinse solution substantially lacking soluble, labelled or epitope-tagged CA (or CR), and thus retained on the capture surface and detected by suitable means, can be identified as candidate ligands.
In a variation, the CA (or CR) can be immobilized on the capture surface and the LBD can be labelled or epitope-tagged. Epitope-tagged proteins can generally be detected by immunochemical methods using at least one antibody species that is specifically reactive with the epitope.
In a variation, the LBD species (or a multiplicity thereof) is immobilized on the capture surface and the soluble, labelled CA and/or CR species (or a multiplicity of species thereof) can be used to identify ligand-induced binding interactions or ligand-dependent relief of binding interactions between an LBD and a CA or CR (or multiple combinations thereof). In such variations, it is usually preferable to employ distinctive labels or epitope-tags for each species of CA and/or CR, which can provide a basis for discrimination of which specie(s) of CA or CR bind to an LBD species (or a collection of LBD species)based upon unique detection of each label or tag on the capture surface. Vice versa, a CA or CR (or multiple species thereof) can be immobilized on the capture surface and multiple species of uniquely labeled or tagged LBDs may be used. In each case, a test agent can be evaluated for its ability to produce a concentration-dependent binding between LBD and CA or CR species and compared to a parallel reaction lacking agent and/or to a parallel reaction lacking agent and containing a known ligand, either agonist or antagonist. Variations which employ multiple species of LBD are termed xe2x80x9cLBD multiplexedxe2x80x9d and variations which employ multiple species of CA or CR are termed xe2x80x9cCA multiplexedxe2x80x9d or xe2x80x9cCR multiplexedxe2x80x9d, respectively. Test agents are categorized based on their effect as an agonist, antagonist, or lack of such effect with respect to each combination of LBD and CA or CR. Concentration dependence and EC50 or IC50 values can be additional parameters for categorization of a test agent as an agonist or antagonist with regard to a particular end point.
The invention employs several formats, such as those just described. These formats can be employed in parallel to provide a multiplexed format assay. A multiplexed format assay of the invention comprises at least two of the following: (1) positive hybrid system:coactivator, (2) positive hybrid system:corepressor, (3) reverse hybrid system:coactivator, (4) reverse hybrid system;corepressor, (5) direct interaction assay, or (6) other art-know assay for identifying and/or quantifying ligand efficacy and/or potency as an antagonist or agonist of nuclear receptors. A preferred multiplex assay is a combination of (1) and (5). Additionally, an individual type of format can be multiplexed with regard to LBD species employed and/or CA or CR species employed (e.g., a positive hybrid system with an estrogen receptor LBD and a second positive hybrid system with a thyroid hormone receptor LBD). Combinations of format-multiplexed and LBD-multiplexed, CA-multiplexed, and/or CR-multiplexed systems and methods can be used in an individual method embodiment to enhance sensitivity and/or selectivity of identification of agonists and antagonists of nuclear receptors.
In an aspect, the invention provides a method for identifying a candidate pharmaceutical agent from a library of test agents, wherein the candidate pharmaceutical agent has a desired biological effect profile (xe2x80x9cbioeffect fingerprintxe2x80x9d). The method comprises: (1) performing n (where n is a number greater than 2, preferably greater than 3, and less than 10 billion) distinct assays of the invention individually using each discrete test agent (which may be a mixture) of the library so as to obtain for each individual test agent measurements of at least n biological effects as detected as a ligand-induced conformational change or a binding interaction change in a discrete assay, (2) for each biological effect detected, assigning a score value (binary or quantitative) based upon the detection (or lack thereof) of a ligand-induced conformational change or binding interaction in each assay, separately, to generate a score matrix (xe2x80x9cbioeffect fingerprintxe2x80x9d) for each agent, and (3) to compare each agent""s score matrix to an equivalent (i.e., representing the same assays under similar conditions) score matrix for one or more predetermined agonist(s) and/or antagonist(s), and thereby identify agents having score matrices substantially similar to the score matrix(ces) of said predetermined agonist(s) or antagonist(s). The substantial identity determination may be made on the basis of the general knowledge and experience of a skilled practitioner in the art, and/or may made on the basis of a rank-ordering of matrix similarity (i.e., total number of matches), and/or may be made by electronic computation using a trained neural network implementation (e.g., BrainMaker running on a Windows platform trained with data from a plurality of predetermined agonists and/or antagonists in similar assays), or by other means. This method provides identification of novel receptor ligands and their characterization based on bioeffect measurements made using in vitro assays and/or cell culture assays. In general, the predictive value of biological effects in vivo of such a multi-parameter method increase with score matrix size (i.e., score matrixes having at least 5 cells or elements, each representing a distinct assay type, are preferred).
In an aspect, the invention also provides novel polypeptides comprising a binding amino acid sequence that is: (1) non-naturally occurring in a nuclear protein, and (2) predetermined to bind to a nuclear receptor, typically by interaction with a binding interface of a nuclear receptor at which naturally-occurring coactivators and/or corepressors bind. These polypeptides and compositions thereof are candidate antagonists for coactivator or corepressor binding (e.g., competitive inhibitors), can be employed as a CA portion of a CA-TRX fusion or as a CR portion of a CR-TRX fusion for use in assays of the invention, such uses can include commercial sale as reagents, and for other uses apparent to those skilled in the art. Such binding amino acid sequences generally comprise a LXXLL (SEQ ID NO:1) motif (wherein L is leucine and X is any of the conventional amino acids) and include, with regard to the LBD of the human estrogen receptor xcex2, the sequences shown in FIG. 19 (SEQ ID NOS:23-40). The novel polypeptides are generally from 5 to 50,000 amino acids long, most usually from 10 to 500 amino acids long, and may comprise one or multiple species of such binding amino acid sequences, which may be repeated, and the remainder of the polypeptide may contain other naturally occuring sequences (e.g., fusions to known proteins) and/or may contain random, pseudorandom, or defined sequence kernal amino acid sequence(s). Peptidomimetics having structural homology to the novel binding sequences are also provided according to methods known in the art; such peptidomimetics or constrained peptides can provide novel therapeutic drugs to provide agonism or antagonism of one or more nuclear receptor ligands.
In an aspect, the nuclear receptor signal transduction system may comprise a transgenic nonhuman animal having all or some somatic cells which are reporter cells. In this aspect, the transgenic nonhuman animal may be adminstered a test agent which may act indirectly (i.e, other than by binding to the LBD) or as an LBD ligand. Detection of reporter expression at a statistically-significant level in said one or more somatic cell types indicates agonist activity of the test agent. In an embodiment, lymphocytes of said transgenic animal are reporter cells and are collected from said transgenic animal following administration of a test agent to the transgenic animal and a suitable incubation period; the relative abundance of lymphocytes expressing the reporter protein as referenced to lymphocytes from an untreated transgenic animal under similar conditions except lacking administration of the test agent serves to identify the agonistic or antagonistic efficacy, if any, of the test compound for activation of the predetermined nuclear receptor in vivo.
Such compositions, reporter host cells, transgenic nonhuman animals, and kits find use as commercially marketable drug development reagents, sensitive diagnostic detection systems for environmental ligands (e.g., to assay polycyclic aromatic hydrocarbons in a sample by detecting their agonistic action on the Ah nuclear receptor and as reagents to titrate biopotency of a nuclear receptor ligand for preparation of pharmaceutical formulations.
Other features and advantages of the invention will be apparent from the following description of the drawings, preferred embodiments of the invention, the examples, and the claims.